WO2021217824A1 - Method for preparing natural flavone selenium - Google Patents

Abstract

Disclosed in the present application is a method for preparing natural flavone selenium. The method comprises the following steps: (1) adding a flavone compound into hot water, uniformly stirring the mixture, and then adjusting same to be alkaline; (2) adding a selenium dioxide aqueous solution and stirring the mixture; (3) adjusting the pH value of the mixture to 3.0-5.5 while cooling same to 20°C-30°C, and uniformly stirring same; and (4) lyophilizing, crushing, and uniformly mixing same to obtain a product. The method has the advantages that natural flavone selenium with a high selenium content, high safety, and tumor resistance is obtained; no organic solvent is used in the preparation process so that pollution is reduced and the operation is safe; water is used as a solvent and basically no waste gas, waste water and waste residues are generated; and the operation is simple.

Description

A method for preparing natural flavone selenium Technical field

This application relates to the field of medical technology, in particular to a method for preparing natural flavone selenium.

Background technique

Flavonoids are widely distributed in various plants, and have anti-tumor, anti-virus, anti-oxidation, scavenging free radicals, strengthening immunity, protecting the cardiovascular system, lowering blood sugar, delaying aging, antibacterial, anti-inflammatory, and regulating the fragility and penetration of capillary Sex and other effects. In recent years, a large number of research results indicate that most of flavonoids play a role in synergy with essential elements of life.

Selenium is an essential trace element for the human body. It has various biological activities such as anti-oxidation, anti-cancer, anti-cancer, protection of bone marrow hematopoiesis, and anti-aging. It also has detoxification effects on some heavy metal elements (such as mercury, arsenic, silver, etc.). There are two sources of selenium in human body, one is inorganic selenium and the other is organic selenium. Inorganic selenium generally refers to sodium selenite and sodium selenate, which are relatively toxic and difficult to be absorbed, and are not suitable for human and animal use. Organic selenium includes simple selenoether, diselenide, and organic selenium oxides. Compared with inorganic selenium, organic selenium has the characteristics of high bioavailability, strong biological activity, low toxicity, and low environmental pollution. Organic selenium compounds synthesized in recent years, such as selenized chitosan, selenoproteins, selenium polysaccharides, selenized carrageenan, selenized tea polyphenols and other selenium small molecules or biological macromolecular compounds are used in anti-tumor and cardiovascular treatment The biological activities of diseases, anti-aging and improving the body's immunity are significantly higher than the corresponding compounds without selenization. Plant active selenium is a source of selenium allowed for use by humans and animals. The use of selenium's unique chemical and biological properties to synthesize organic selenium compounds with high biological activity and low toxicity has been one of the current hot spots in drug research.

Summary of the invention

This application provides a method for preparing natural flavone selenium.

This application adopts the following technical solutions:

A method for preparing natural flavone selenium, characterized in that, the method comprises the following steps:

(1) Add flavonoids to hot water, stir evenly and adjust to alkaline;

(2) Add selenium dioxide aqueous solution and stir;

(3) Adjust the pH to 3.0-5.5 when cooled to 20-30°C, and stir evenly;

(4) Freeze-drying, crushing and mixing to obtain the product.

Further, in step (1), the flavonoid compound is tea polyphenol, dihydromyricetin, chrysin, baicalein, myricetin, etc.

Further, in step (1), after adding the flavonoids to the hot water, it is heated for 30-60 minutes.

Further, in step (1), the pH is adjusted to 8-11.

Further, in step (2), the stirring time is 30-90 min.

Further, in step (3), the pH is adjusted using hydrochloric acid solution, glacial acetic acid solution, sulfuric acid solution, hydrobromic acid solution, or the like.

Further, the mass ratio of flavonoids to selenium dioxide is 1:0.1-10.

The beneficial effects of this application are as follows:

(1) The method of the present application obtains a natural flavonoid selenium with high selenium content, high safety, and anti-tumor.

(2) No organic solvents are used in the preparation process, reducing pollution and safe operation.

(3) The method of this application uses water as the solvent, and basically no three wastes are generated.

(4) The operation of the method of this application is very simple.

Description of the drawings

Figure 1 shows the fingerprint of natural flavonoids and selenium.

Figure 2 shows the fingerprint of natural flavonoids and selenium.

Figure 3 shows the results of thermogravimetric analysis of natural flavonoids and selenium.

Detailed ways

In order to make the purpose, technical solutions, and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely in conjunction with specific embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of this application.

Example 1: Preparation test

(1) Add dihydromyricetin (450g) to hot water, heat for 30min, stir evenly, adjust the pH to 11 with sodium hydroxide solution;

(2) Add selenium dioxide (100g) aqueous solution and stir;

(3) Use hydrochloric acid solution to adjust the pH to 4.0 when cooled to 20-30°C, and stir evenly;

(4) Freeze-drying, crushing and mixing to obtain the product.

Example 2: Preparation test

(1) Add dihydromyricetin (450g) to hot water, heat for 30min, stir evenly, adjust the pH to 11 with sodium hydroxide solution;

(2) Add selenium dioxide (150g) aqueous solution and stir;

(3) Use hydrochloric acid solution to adjust the pH to 4.0 when cooled to 20-30°C, and stir evenly;

(4) Freeze-drying, crushing and mixing to obtain the product.

Example 3: Preparation test

(1) Add dihydromyricetin (450g) to hot water, heat for 30min, stir evenly, adjust the pH to 11 with sodium hydroxide solution;

(2) Add selenium dioxide (200g) aqueous solution and stir;

(3) Use hydrochloric acid solution to adjust the pH to 4.0 when cooled to 20-30°C, and stir evenly;

(4) Freeze-drying, crushing and mixing to obtain the product.

Example 4: Toxicity test of natural flavone selenium on ICR mice

(1) Reagents and materials

Sterilized water for injection

Source: Guangdong Aixide Pharmaceutical Co., Ltd.

Properties: colorless transparent liquid

Lot number: 180416205

Specification: 500ml/bottle

Storage conditions: RT

(2) Drug preparation

1000mg reagent is prepared into 4ml solution with sterile water for injection

(3) Experimental animals

Breeds and strains: ICR mice

Level: SPF level

Gender: half male and half

Source: Shanghai Sipuer-Bikai Laboratory Animal Co., Ltd.

Laboratory animal quality certificate number: 2008001635879

Laboratory animal production license number: SCXK (Shanghai) 2018-0006

Number of animals: order 6 males and 6 males each, a total of 12 are used for the experiment

Animal age at the beginning of the experiment: 6～10w

Animal weight at the beginning of the experiment: 20g±20%

Adaptation time: 3 days, the same feeding conditions as the experiment

Animal numbering method: Each squirrel cage is equipped with an identification card with information such as experiment number, experiment group, experimenter's name, animal breed and gender, and the mouse is marked with a line at the base of the tail.

(4) Environment

The environment of the animal room is maintained at a temperature of 23±2°C, a humidity of 40-70%, and alternating light and dark for 12 hours. The animals are reared with 2 to 4 animals in each cage, and the bedding is changed twice a week (corncob bedding, Suzhou Daichuan Trading Co., Ltd.).

(5) Food and drinking water

During the adaptation period, the SPF growth and reproduction feed for rats and mice was fed with Co ⁶⁰ sterilized and purchased from Beijing Keyao Xieli Feed Co., Ltd. High-pressure sterilized filtered water was used for experimental animal water.

(6) Animal selection and eating

The animals used in the experiment will remain healthy. The animals eat and drink freely during the experiment.

(7) Test method

7.1 Grouping

After the adaptation period, the 12 experimental animals were divided into 2 groups, half male and half male, and the solvent control or test substance was given by oral gavage according to their body weight:

7.2 Clinical symptoms

Observe the clinical symptoms of the animals after the administration, observe the cage for 1 week, and have abnormal records.

7.3 Weight

Weigh the animals before administration.

7.4 End of test

At the end of the experiment, the animals were euthanized _{by inhalation of excessive CO 2.}

(8) Statistics

The experimental data are expressed as Mean±SEM; the data between the two groups were analyzed by unpaired t-test, and p<0.05 was considered as a significant difference.

(9) Results

Clinical observation and mortality

Animals in the Vehicle group and AQ-C-a 5000mg/kg group did not show any obvious abnormalities during the experiment. The mortality rate is 0%.

Example 5: In vitro tumor cell experiment of natural flavone selenium

(1) Experimental equipment

(2) Reagent

(3) Experimental cells

(4) Experimental method

4.1 Cell culture

4.1.1 Observe the cell status under a microscope, the cells are free of contamination, and the cell confluence is about 90%;

4.1.2 Discard the supernatant, add 5mL PBS to each dish and wash again, add 3mL trypsin, digest for 3min; or change the solution 5mL;

4.1.3 Gently blow the cells, collect the cells in a centrifuge tube, and centrifuge at 1000 rpm for 5 min;

4.1.4 Discard the supernatant, resuspend part of the cells in fresh medium, and transfer them to a new cell culture dish to expand the culture, with a passaging ratio of 3:8;

4.1.5 Continue to pass down and further expand the culture according to the experimental plan, and the confluence is about 90%.

4.2 Drug preparation

Weigh 30.28mg, dissolve it with 0.4ml DMSO to obtain the mother liquor, and dilute to working concentration.

4.3 Dosing and testing of CCK-8 paving

4.3.1 Cell seeding plate: Digest the logarithmic growth phase cells with trypsin to prepare a cell suspension, inoculate 3000-5000 cells per well in a 96-well plate, add 100μl per well, and place in CO ₂ (5%) Incubate overnight at 37°C in an incubator to adhere to the wall, and fill the edge holes with sterile PBS.

4.3.2 Add different concentrations of compounds to a 96-well plate, and set 3 replicates for each sample concentration.

4.3.3 CCK-8 reaction: add 10μl of CCK-8 solution to all wells, tap the culture plate gently to mix, and incubate in the incubator for 2 hours.

4.3.4 Measuring the absorbance value: Use a microplate reader to measure the 450nm absorbance value, and calculate the inhibitory rate of the drug on the cells according to the formula.

(5) Data analysis

Use Graghpad-prism5.0 as a graph to calculate the IC50 value of each drug in each cell at the corresponding time point.

(6) Experimental results

Example 6: Fingerprint of natural flavone selenium

(1) Instrument

Dual ternary high performance liquid chromatograph, U3000-CAD

Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicines, Version 2012.130723

(2) Reagent

Methanol, chromatographic grade

Acetic acid, chromatography grade

Ultrapure water, 18.2MΩ.cm@25℃

(3) Chromatographic conditions

(4) Solution preparation

The preparation of 50% acetonitrile solution: Take 1000ml of acetonitrile, add 1000ml of purified water, and mix well to get it.

Preparation of test solution: Pipette about 1ml of the central control sample solution, place it in a 50ml volumetric flask, add 50% acetonitrile solution and dilute to the mark, sonicate for 10min, filter, shake, and use as the test solution.

(5) Test

Precisely draw 10μl of the test solution into the dual ternary high performance liquid chromatograph, measure, record the chromatogram, and get it.

The peak height of the highest peak in the chromatogram should be 50-300pA. If the highest peak of the test solution exceeds this range, adjust the dilution factor of the 4.2 central control sample.

According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the similarity between the fingerprints of the test product and the control fingerprints is calculated.

The similarities between the natural flavonoid selenium compounds prepared by the examples of this application are all greater than 0.95.

Example 7: Natural flavonoids, selenium and selenium content

(1) Instrument

Electronic balance, XPE205

Microwave Digestion Apparatus, MWD-520

Atomic absorption spectrophotometer, AA-6880

(2) Reagent

Nitric acid, premium grade pure

Ultrapure water, 18.2MΩ.cm@25℃

(3) Standard solution

Se standard solution, 1000mg/L

(4) Solution preparation

4.1 Preparation of blank solution

Take 7ml of nitric acid, place it in the microwave digestion inner tank, place it for 15 minutes to pre-digest, screw the lid tightly and perform microwave digestion. The digestion parameters are shown in Table 1. After the digestion is complete, take out the digestion tank after cooling, and transfer the digestion solution to 50ml In the volumetric flask, make the volume up to the mark with water, then draw 2ml to a 50ml volumetric flask, add water to the volume, shake well and set aside.

4.2 Preparation of working standard solution

Precisely pipette 0ml, 0.5ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml of selenium standard solution (1000mg/L) into a 100ml volumetric flask respectively, dilute to the mark with 2% nitric acid, and shake well. The concentration of the working standard solution is 0mg/L, 5mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L.

4.3 Preparation of test solution

Take about 200mg of the test product, accurately weigh it, place it in the microwave digestion inner tank, add 7ml of nitric acid, leave it for 15 minutes to pre-digest, tighten the lid, and perform microwave digestion. The digestion parameters are shown in Table 1. After the digestion is complete, after cooling Take out the digestion tank, transfer the sample digestion solution to a 50ml volumetric flask, dilute to the mark with water, then accurately transfer 2ml of the above solution to a 50ml volumetric flask, add water to dilute to the mark, shake well for use.

Table 1 Microwave digestion parameters

step	Digestion temperature (℃)	Heating time (s)	Constant temperature time (s)
1	120	300	300

2	150	300	600
3	190	300	1200

(5) Determination

Determination of working standard solution: Take the working standard solution and determine it according to flame atomic absorption spectrometry (Four General Principles of Chinese Pharmacopoeia). The concentration is measured from low to high, and the absorbance value is recorded. The concentration value of the standard solution and its corresponding absorbance value are used to calculate the regression Equation, the correlation coefficient r shall not be less than 0.99.

Determination of the test solution: take the test solution, adjust the zero with the blank control solution, and record the absorbance value of the test solution. The test data is processed by the regression equation calculation method, and the result is calculated by the following formula.

(6) Calculation

In the formula: C is the concentration of the test solution after blank correction, in mg/L

V is the constant volume of the digestion solution of the test substance, the unit is L

f is the dilution factor of the test solution

W is the sampling amount of the test product, calculated as dry product, the unit is g

(7) Results

The selenium content of natural flavone selenium was 15.1%.

Example 8: Thermogravimetric analysis of natural flavonoids and selenium

Raise temperature to 150.00°C at a rate of 10.00°C/min;

Keep isothermal for 20 minutes;

The temperature is increased to 300.00°C at a rate of 10.00°C/min.

The result is shown in Figure 3.

The above descriptions are only examples of the present application, and are not used to limit the present application. For those skilled in the art, this application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included in the scope of the claims of this application.

Claims (10)

1. A method for preparing natural flavone selenium, characterized in that, the method comprises the following steps:

   (1) Add flavonoids to hot water, stir evenly and adjust to alkaline;

   (2) Add selenium dioxide aqueous solution and stir;

   (3) Adjust the pH to 3.0-5.5 when cooled to 20-30°C, and stir evenly;

   (4) Freeze-drying, crushing and mixing to obtain the product.
2. The method of claim 1, wherein in step (1), the flavonoid compound is tea polyphenol, dihydromyricetin, chrysin, baicalein or myricetin.
3. The method according to claim 1, wherein in step (1), after adding the flavonoids to the hot water, heating is performed for 30-60 minutes.
4. The method of claim 1, wherein in step (1), the pH is adjusted to 8-11.
5. The method according to claim 1, wherein in step (2), the stirring time is 30-90 min.
6. The method according to claim 1, wherein in step (3), the pH is adjusted using a hydrochloric acid solution, a glacial acetic acid solution, a sulfuric acid solution, or a hydrobromic acid solution.
7. The method according to claim 1, wherein the mass ratio of flavonoids to selenium dioxide is 1:0.1-10.
8. The natural flavone selenium prepared by the method of any one of claims 1-7.
9. The use of natural flavone selenium as claimed in claim 8 in the preparation of anti-tumor drugs.
10. The application according to claim 9, wherein the tumor is liver cancer, lung cancer, colorectal cancer, cervical cancer, gastric cancer or prostate cancer.

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